Chapter 3: Targeting Human Telomerase via mRNA Display
C.1 Introduction
A p p e n d i x C
PROBING THE INTERACTIONS BETWEEN HUMAN TELOMERASE RNA AND THE HUMAN TELOMERASE REVERSE TRANSCRIPTASE RNA-
BINDING DOMAIN
C.2 The Human Telomerase Reverse Transcriptase RNA-Binding Domain
Our lab has created a construct for the expression of the hTERT RNA-binding domain (hTERT-288), the sequence between amino acid residues 326 and 613 (hTERT numbering). Using appropriate primers (hTERT288-5: 5’-CCCGGATCCGCCGAGACCCA AGC-3’; and hTERT288-3: 5’-CGTGAATTCTTATCAGTGGTGGTGATCATCGTGCCT GGCTTCCCGATGC-3’) the hTERT288 sequence was PCR amplified in a 50 μL reaction containing PCR buffer (10 mM Tris-Cl, pH 9.0; 50 mM KCl, 2 mM MgCl2; 0.1% Triton X- 100; 0.2 mM each of dATP, dGTP, dCTP, and dTTP), 40 pmol each primer, 1 ng vector containing the hTERT sequence (generously donated by the Weinberg lab), and 1 μL TAQ.
Reactions were heated to 94 oC, followed by 29 cycles of 94 oC for 30 seconds, 60 oC for 1 minute, and 72 oC for three minutes. The PCR product was cleaned via the QiaQuick PCR Purification Kit (Qiagen) and cloned into the pGEX-2T vector (Amersham) using the BamH I and EcoR I sites. We added an N-terminal glutathione-s-transferase (GST) for purification and solubility purposes, as well as a C-terminal histidine (his) tag for purification purposes. In order to accommodate the rare codons found in the hTERT sequence, the vector was transformed into the Rosetta BL21-DE3 line, which supplies rare tRNA codons.
The protein was expressed in E. coli. A 5 mL LB + 50 μg/mL ampicillin was grown overnight at 37 oC with shaking. The starter culture was then added to 1 L of fresh LB and allowed to grow at 37 oC with shaking for ~2 hours or until the OD600 reached ~0.5. IPTG (1 mM final concentration) was added for induction of protein expression. The protein was expressed at 37 oC for 4 hours with shaking. While expression in E. coli was successful, the resulting protein was insoluble. Strategies to increase solubility, by utilizing an N-terminal
ubiquitin tag or inducing slower expression by reducing the temperature from 37 to 30 oC and using less IPTG, were generally unsuccessful in producing soluble protein. Denaturing purification followed by refolding was also unsuccessful in producing a functional hTERT- 288.
We then decided to express hTERT-288 in insect cells, since this approach has proven successful in producing purified hTERT with in vitro activity (Masutomi et al. 2000; Mikuni et al. 2002). The protein expression was performed by Dr. Peter Snow (Caltech Protein Expression Center) according to established protocols (Masutomi et al. 2000; Mikuni et al.
2002) using the Baculovirus Expression Vector System. The protein was purified via the GST tag under native conditions. The cells were lysed by resuspending the cell pellet in 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 1% NP-40, 5 mM EDTA, and protease inhibitors (Roche) followed by sonication on ice. The lysed cells were spun down and the lysate applied to a prewashed GST column. The protein was washed with ten column volumes of lysis buffer followed by washing with thirty column volumes of 20 mM Tris-Cl, pH 8.0, and 100 mM NaCl. The protein was then eluted in 50 mM Tris-Cl, pH 8.0, and 20 mM glutathione. Since GST is expressed in the insect cells, endogenous GST was purified from the lysate along with the hTERT-288 construct (figure C.1). Efforts to utilize the His tag for a second purification step were unsuccessful, possibly due to conformational constraints that make the tag inaccessible in the native protein (data not shown). The GST and His tags were not cleaved since the amount of soluble protein purified via the GST tag was low, and further cleavage and purification steps led to an amount of protein insufficient for biochemical analysis.
GST
GST
hTERT ladder
hTERT 25kD
50kD 75kD
Figure C.1. SDS-PAGE gel of the hTERT-288 protein, purified by the GST tag. hTERT-288 was expressed as a GST fusion, resulting in a protein that is ~55 kD.
C.3 Mobility-Shift Assay between the hTERT-288 Protein and hTR
Preliminary results indicate that the hTERT-288 construct binds to several hTR constructs. The constructs were constructed in a manner similar to the construction of RNA33-147 from chapter 2. Using appropriate PCR primers, RNA208-451, RNA163-330, RNA208-330, and RNA208-360 were PCR amplified in 100 μL reactions from a pUC19 vector containing the DNA sequence encoding for hTR-451 (the complete wild-type hTR sequence). The PCR products were phenol extracted, ethanol precipitated, and digested with BsmB I to cut the vector and generate appropriate ends for run-off transcription. The DNA was then phenol extracted, ethanol precipitated, and dried to pellets. The pellets were then used in 1 mL transcriptions using T7 RNA polymerase. The RNA was gel purified, electroeluted into 0.5X TBE, and ethanol precipitated. The resulting pellets were resuspended in ddH20 and the concentrations determined by UV spectroscopy and the biopolymer calculator developed by the Schepartz lab (Palmer 1998).
Mobility-shift experiments were performed using the partially purified hTERT-288 construct (figure C.2; figure C.3). The reactions contained 5 nM 32P-end labeled RNA, 0.2 μg or 1 μg hTERT-288, 10 mM Tris-Cl, pH 8.0, 1 mM DTT, 50 mM NaCl, and 10% glycerol.
The reactions were assembled without the protein, annealed at 90 oC for 90 seconds and set on ice for two minutes. After addition of protein, the reactions were allowed to sit on ice for 30 minutes before loading to a 6% native gel run in 0.5X TBE at 4 oC. The gel was run at 2W for 2 hours and 40 minutes, dried, and exposed to a PhosphorImager screen. The screen was scanned on a Storm860, and the data analyzed by ImageQuant.
Figure C.2. Competition binding assay between RNA208-451 and increasing concentrations of hTR-451 shows that RNA208-451 and hTR-451 bind to the hTERT RNA-binding domain in a similar manner. Each lane contains 5 nM RNA208-451 and varying concentrations of hTR-451. Lanes 2-6 contain 0.2 μg hTERT-288 protein. Lane 2=no hTR-451; lane 3=5 nM hTR-451; lane 4=25 nM hTR-451; lane 5=50 nM hTR-451; lane 6=500 nM hTR-451.
163-330 208-330 208-360
free free
complex complex complex free
1 2 3 4 1 2 3 4 1 2 3 4 labeled RNA:
Figure C.3. Mobility-shift assay between hTERT-288 and three hTR constructs. The labeled RNAs (RNA160-330, RNA208-330, and RNA208-360) are CR4/CR5-containing constructs.
1=no protein; 2=GST only; 3=0.2 μg hTERT-288; 4=1 μg hTERT-288. RNA208-330 is sufficient for binding to the hTERT RNA-binding domain.
The hTERT-288 protein displays the ability to bind functionally important regions of hTR. All the constructs tested contain the catalytically critical CR4/CR5 domain. RNA208- 451 shifts in the presence of hTERT-288 and can be competed off by hTR-451 (figure C.2).
Using the mobility-shift assay, the approximate Kd of the interaction between RNA208-451 and hTERT-288 is 2 μM (data not shown). This value is on the order of previously reported values for association of hTERT and hTR (Xia et al. 2000). As figure C.3 shows, GST does not result in a shift of the labeled RNAs, indicating that the GST tag is likely not participating in the interaction with the RNAs. The data also show that RNA163-330, RNA208-360, and RNA208-330 shift in the presence of hTERT-288 indicating RNA208-330 is sufficient for binding to the RNA-binding domain of hTERT.
Though these results are preliminary, the 123 nucleotide RNA/288 amino acid protein association presented here is a much smaller system compared to the wild-type 451-nucleotide hTR/1132 amino acid hTERT association. A smaller system may be more amenable to future biochemical and biophysical analysis. Additionally, hTERT-288 can be utilized in RNA selection experiments that involve constructing a library of random nucleotides to isolate sequences that bind with high affinity to the hTERT RNA-binding domain. The resulting RNA ligands will provide information about the structural and sequence requirements of hTERT for its RNA partner as well as potentially provide leads for the development of anticancer therapies.